Residues from Homologous Transmembrane Helices 4 and 10 Are Critical for P-Glycoprotein (ABCB1)-Mediated Drug Transport
Abstract
:Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Chemicals and Antibodies
2.2. Cell Line and Culture Conditions
2.3. BacMam Baculovirus Transduction of HeLa-S3 Cells and Cell Surface Expression of the Mutants
2.4. Transport of Fluorescent Substrates
2.5. Cytotoxicity Assays
2.6. Preparation of Membrane Vesicles of High Five Insect Cells
2.7. SDS-PAGE and Western Blotting
2.8. ATPase Activity
2.9. Molecular Modeling and Electrostatic Surface Potential
2.10. Molecular Dynamics (MD) Simulations
2.11. Statistical Analysis
3. Results
3.1. Rationale for Studying Homologous Transmembrane Helices TMH4 and TMH10 and the Selection of Residues to Generate Mutants
3.2. Both TMH4-7A and TMH10-7A Mutants Have a Similar Level of Expression as WT P-gp
3.3. Seven Alanine Substitutions in TMHs 4 and 10 Do Not Have Any Significant Effect on the Transport Function
3.4. The Expression and Overall Conformation of the TMH4,10-14A Mutant Is the Same as WT P-gp
3.5. TMH4,10-14A Mutant Failed to Transport All the Tested Substrates
3.6. Although the TMH4,10-14A Mutant Exhibits Basal ATPase Activity the Same as That of WT P-gp, Substrates Fail to Stimulate It
3.7. Mutations in TMH4,10-14A Do Not Significantly Affect the Electrostatic Surface Potential in the Drug-Binding Cavity
3.8. MD Simulation of TMHs 4 and 10 of the TMH4,10-14A Mutant and WT P-gp
3.9. Omitting Three Alanine Substitutions in TMH 4 in the TMH4,10-14A Mutant Recovers the Transport of a Few Substrates
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Substrates | TMH4,10-14A | TMH4,10-11A | TMH4-7A | TMH10-7A |
---|---|---|---|---|
BD-CsA | 33 ± 3 | 3 ± 0.6 | 89 ± 3 | 85 ± 2 |
BD-EDA | 22 ± 5 | 66 ± 6 | 93 ± 67 | 46 ± 2 |
BD-Prazosin | 2 ± 0.6 | 54 ± 4 | 40 ± 10 | 18 ± 13 |
BD-Verapamil | ND | 3 ± 0.6 | 26 ± 12 | 3 ± 2 |
Calcein-AM | 23 ± 9 | 58 ± 9 | 94 ± 4 | 97 ± 3 |
Daunorubicin | 21 ± 1 | 34 ± 2 | 64 ± 6 | 54 ± 9 |
Flutax-1 | ND | 4 ± 2 | 98 ± 4 | 92 ± 7 |
LDS 751 | 31 ± 8 | 43 ± 6 | 89 ± 8 | 91 ± 8 |
NBD-CsA | 3 ± 1 | 4 ± 1 | 95 ± 9 | 94 ± 11 |
R6 | 13 ± 11 | 6 ± 2 | 54 ± 6 | 13 ± 1 |
R6G | 43 ± 3 | 78 ± 3 | 90 ± 1 | 95 ± 1 |
Rh123 | 3 ± 0.58 | 28 ± 5 | 93 ± 3 | 90 ± 3 |
Rhod-2 AM | ND | 5 ± 2 | 96 ± 6 | 96 ± 5 |
TMRE | 26 ± 5 | 85 ± 1 | 100 ± 0 | 98 ± 3 |
X-Rhod-1 AM | 4 ± 1 | 4 ± 1 | 96 ± 6 | 99 ± 1 |
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Rahman, H.; Ware, M.J.; Sajid, A.; Lusvarghi, S.; Durell, S.R.; Ambudkar, S.V. Residues from Homologous Transmembrane Helices 4 and 10 Are Critical for P-Glycoprotein (ABCB1)-Mediated Drug Transport. Cancers 2023, 15, 3459. https://doi.org/10.3390/cancers15133459
Rahman H, Ware MJ, Sajid A, Lusvarghi S, Durell SR, Ambudkar SV. Residues from Homologous Transmembrane Helices 4 and 10 Are Critical for P-Glycoprotein (ABCB1)-Mediated Drug Transport. Cancers. 2023; 15(13):3459. https://doi.org/10.3390/cancers15133459
Chicago/Turabian StyleRahman, Hadiar, Mark J. Ware, Andaleeb Sajid, Sabrina Lusvarghi, Stewart R. Durell, and Suresh V. Ambudkar. 2023. "Residues from Homologous Transmembrane Helices 4 and 10 Are Critical for P-Glycoprotein (ABCB1)-Mediated Drug Transport" Cancers 15, no. 13: 3459. https://doi.org/10.3390/cancers15133459